CA2034734A1

CA2034734A1 - Amperometric detection cell

Info

Publication number: CA2034734A1
Application number: CA002034734A
Authority: CA
Inventors: Mark L. Bowers; David Buttaro; W. Michael Krebs
Original assignee: ESA Inc
Current assignee: Magellan Diagnostics Inc
Priority date: 1990-03-02
Filing date: 1991-01-22
Publication date: 1991-09-03
Also published as: JPH0772135A; DE69106185T2; ATE116437T1; EP0447288B1; DE69106185D1; EP0447288A1

Abstract

IMPROVED AMPEROMETRIC DETECTION CELL

ABSTRACT OF THE DISCLOSURE

An improved electrochemical detection cell comprising a palladium reference electrode.

Description

1 IMPROV~D AMPEROM~TRIC DBTECTION CELL
, 2 The present invention relates to a new electrochemical 3 detector device and more particularly, to a new 4 amperometric detector cell for qualitatively and quantitatively testing electroactive materials in 6 solution. The invention has particular utility in 7 connection with liquid chromatography separations and 8 detection of amino acids and carbohydrates and will be 9 described in connection with such use, although other uses are contemplated.
11 With the burgeoning interest in genetic engineering 12 and biotechnology, the determination of amino acids for 13 protein sequencing and analysis has become increasingly 14 important.
Recent progress in amino acid determinations can be 16 attributed, in part, to technological advances in liquid 17 chromatography and chromatographic detectors. Separations 18 of amino acids and their derivatives in liquid 19 chromatography (LC) are readily achieved by using reversed-phase stationary phases and ion exchangers. For 21 the separation of complex mixtures, gradient elution 22 chromatography is essential.
23 In recent years, electrochemical detection with liquid 24 chromatography has gained prominence as a sensitive and selective detection technique for electroactive groups.
26 Amino acids and carbohydrates generally have not been 27 considered to be electroactive. Direct anodic detection 28 at constant applied potential (DC) can occur by catalytic 29 mechanisms on certain transition-metal oxides, e.g. nickel and copper. However, aatalytic DC detec,tions on nobel-31 metal electrodes is accompanied by 108s of electrode 32 activity with rapid decay of analytical sensitivity.
33 Pulsed amperometria detection (PAD) and pu,lsed 34 coulometric detection (PCD) following liquid chromatography has proven to be selective and sensitive 36 techniques for the determination of alcohols, -2- ~3~

1 polyalcohols, carbohydrates, amino acids, aminoalkanols ; 2 and many inorganic and organic sulfur-containing 3 compounds. PAD uses a triple-step potential waveform to 4 combine amperometric (PAD) detection followed by alternating anodic and cathodic polarizations to clean and 6 reacti~ate the electrode surface whereas PC~ uses three or ~ 7 more potential steps in a wave form to combine coulometric `~ 8 detection and eliminate the undesirable signal due to the 9 electrode itself followed by the cleaning potential. In the detection of amino acids ancl carbohydrates, the 11 waveform exploits the surface-catalyzed oxidation of the 12 amine and alcohol functionalities activated by the 13 formation of nobel metal surface oxides. Sensitivity in 14 PCD is optimum at ca.pH > 11, and postcolumn addition of base may be desired. However, the catalytic nature of PC~
16 for amino acids limits the use of gradient elution 17 chromatography because the base-line signal corresponds to 18 the oxide formation process which is very sensitive to 19 small changes in the mobile phase composition, especially the pH.
21 As report~d by Welch et al ~n their article Comparison 22 of Pulsed Coulometric Detection and Potential-Sweep Pulsed 23 Coulometric Detection for Underivatized Amino Acids in 24 ~iquid Chromatography in Analytical Chemistry, 1989, 61, 555-55g, a major limitation in the electrochemical 26 detection with liquid chromatography is the inability to 27 efficiently couple gradient chromatography with pulsed 28 electrochemical detection. According to nelsh et al prior 29 attempts to use a four-solvent gradient system LC/PCD for the separation of a 17-component hydrolyzate resulted in 31 such a severe base-line shift, as predicted by the cyclic 32 voltammograms, so as to make the chromatograms virtually 33 useless. A major cause for the shifting of the surface 34 oxide background is a change in pH~ While a glass pH
electrode reportedly results in a shift of the reference 36 potential substantially in unison with the pH gradient, _3_ ~3~

1 a comparison of three-gradient solvents by cyclic 2 voltammetry using a pH reference electrode revealed that 3 the anodic waves for oxide formation are nearly 4 superimposed.
Another problem that is not generally recognized with 6 amperometric detectors and especially amperometric 7 detectors used in pulse detection is the large junction 8 potentials and the large uncompensated current-resistance 9 (IR) that is present with the generally used reference electrodes such as the silver/silver chloride and the 11 saturated calomel electrode which are usually contained in 12 glass tubPs and separated from the flow stream by a porous 13 glass or ceramic barrier. These typical reference 14 electrode arrangements can lead to inadequate potential control of the working electrode and place larger demands 16 on the compliance voltage of the potentiostat. These in 17 turn can result in added noise and slower response of the 18 EC detector.
19 We have found that the aforesaid and other problems of the prior art may be obviated by the use of a solid state 21 palladium reference electrode. ~ore particularly, in 22 accordance with the present invention, an amperometric 23 detection cell is provided comprising a three-electrode 24 system consisting of a working electrode of conventional construction, for example, gold, platinum, ~lassy carbon 26 or the like, a counter-electrode of conventional 27 construction, and a solid state palladium reference 28 electrode which is actually driven in response to changes 29 in the test solution. Typically the reference electrode comprises at least one thin wire formed of palladium or 31 palladium oxide, and the counter-electrode comprises a 32 flat plate or foil formed of a metal such as platinum or 33 gold. Alternatively, the counter-electrode may also be 34 formed of palladium or palladium oxide.
For a further understanding of the nature and 36 advantages of the present invention, reference should be - : . . . : .
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1 had to the following detailed description taken in 2 connection with the accompanying clrawings, wherein like 3 numbers denote like parts, and wherein:
4 FIG. 1 i~ a schematic view of one form of liquid chromatography apparatus incorporating an electrochemical 6 detection apparatus in accordance with the present 7 invention;
8 FIG. 2 is a side elevational view, in cross-section, 9 of a preferred form of electrochemical de ector made in accordance with the present invention;
11 FIG. 3 is a cross-sectional view of an electrochemical 12 detection cell of FIG. 2, taken along lines 3-3;
13 FIG. 4 is a cross-sectional view o the 14 electrochemical detection cell of FIG. 2, taken along lines 4-4; and 16 FIG. 5 is a diagrammatic sketch of a preferred form of 17 reference electrode control circuit of the present 18 invention.
19 Referring to FIG. 1, there is illustrated a liquid chromatography apparatus and electrochemical detection 21 apparatus in accordance with the present invention. The 22 illustrated liquid chromatography apparatus includes 23 mobile phase reservoirs 20a...20n coupled through suitable 24 valves a constant volume pump means 22 and an injection valve 24 and sample inlet 26 to the top of a liquid 26 chromatography column indicated generally at 28. In 27 practice, sample materials to be tested are introduced 28 into the chromatography apparatus either by direct 29 injection of microliter amounts of sample material into the chromatography cdlumn 28, e.g. through a syringe at 31 sample inlet 26, or the sample material may be introduced 32 into the chromatography column 28 as a dilute solution of 33 sample material at injection valve 24. Thus, if desired, 34 either injection valve 24 or sample inlet 26 may be omitted from the system. Chromatography column 28 is 36 packed with selected ion exchange resins in bed or powder '' ~ :: . ' ' . ::-' .':

2 g~ 3 ~

1 form. The selection of the mobile phase, and the 2 selection and packing order of the ion exchange resins 3 will depend on the particular separations desired and can 4 readily be determined by one skilled in the art and thus will not be further described herein. The base of 6 chromatography column 28 is coupled via an outlet 30 to a 7 splitter valve 32 which divides the eluant from the 8 chromatography column 28 between a sample collection 9 vessel or waste container 34 and an electrochemical detection apparatus made in accordance with the present 11 invention, and indicated generally at 36.
12 The illustrated chromatography apparatus (other than 13 the electrochemical detection apparatus 36) is 14 conventional and may be of the type described by P.H.
Freeman and W.L. Zielinski, in U.S. Bureau of Standards 16 Technological ~ote Number 589, Page 1, (July 1970 to June 17 1971). Moreover, it should also be noted that the 18 electrochemical detection apparatus 36 of the present 19 in~ention is not limited to use with the particular type of chromatography apparatus illustrated in FIG. 1, which 21 is merely given as exemplary.
22 As mentioned supra, a problem with prior art 23 amperometric detectors not employing pH reference 24 electrodes, is the inability to compensate fully for pH
gradient shifts resulting from the use of different mobile 26 phases. The present invention overcomes the aforesaid and 27 other disadvantages of prior art amperometric detectors by 28 employing a solid state reference electrode formed of 29 palladium or palladium oxide. The use of a solid state palladium reference electrode in a coulometric detection 31 cell is described in U.S. Patent 4,404,065 issued 32 September 13, 1983 to Wayne R. Matson, and assigned to the 33 assi~nee of the present application. However, prior to 34 the present invention, the advantages of employing a solid state palladium reference electrode in an amperometric 36 cell were not recognized.

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' ,', ' 2~ 7?4 1 Referring to FIGS. 2 and 3, electrochemical detection : 2 apparatus 36 comprises an electrochemical detection cell 3 comprising a cylindrical body 38 having a pair of end 4 plates 40 and 42, respectively. Main cell body 38 and plates 40 and 42 comprise short, generally cylindrical 6 plates ormed o a rigid, liquid impervious, electrically-: 7 insulating, chemically inert material such as a synthetic 8 polymeric material, e.g. a ceramic, an unplasticized 9 polyvinyl chloride, a polytetrafluoroethylene fluorocarbon resin, or the like. An internally threaded screw mounting 11 (not shown) is formed in plate 42 for connecting the 12 outlet from chromatography column 28 via conduit 44 to an 13 inlet 46. Inlet 46 communicates with a bore 48 formed in 14 body 38 to the cell thin layer detection flow path indicated generally at 50 n In a similar manner, the 16 reference electrode assembly 52 is threadedly mounted 17 through a internally threaded screw mounting (not shown) 18 in plate 42 and communicates via a bore 54 with the thin 19 Iayer flow path 50. Flow path 50 communicates through a bore 56 formed in the side wall of body 38 to a threaded 21 fitting 58 fitted with an outlet conduit 60.
22 Tha test elec-trode assembly is mounted on plate 40, 23 and comprises a cylindrical spacer member 62 and cap 24 member 64 which are threaded together, and the assembly in turn is threaded into an internally threaded aperture (not 26 shown) in plate 40. The test electrode is mounted in a 27 bore (not shown) which extends through members 62 and 64 28 and through plate 40 to the interior of the electrode 29 assembly. Completing the electrode chemical cell assembly is a rigid spacer member 68 and gasket 70 as will be 31 described in detail hereinafter. Members 62 and 64 and 32 spacer 68 all are formed of a rigid, liquid impervious 33 electrically-insulating, chemically inert material~
34 Gasket 70 preferably is formed of Tef].on. A plurality of bolt holes 70 are formed through end plates 40, 42, spacer 36 68 and gasket 70 and provide entry for bolts, only one of .
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1 which 72 is shown. Bolts 72 align the individual parts of 2 the-electrochemical detection cell and, when anchored with 3 nuts 74, apply pressure to keep the electrochemical 4 detection cell together.
Referring now to FIG. 3, a recess 32 is formed in body 6 38, communicating with inlet 48 and outlet 56. A 0.25 7 millimeter thick palladium foil counter electrode 86 is 8 positioned in the bottom of recess 82, and it is held in 9 place by means of a Teflon gasket 84. A palladium wire reference electrode is threaded through bore 56 with its 11 end mounted flush with counter electrode 81.
12 Referring to FIG. 4, the testing electr~de, which 13 comprises a solid gold wire 90 is positioned with its end 14 surface flush with the surface of spacer 68. A bushing member 92 formed of Teflon or the like, surrounds 16 electrode 90, and maintains electrode 90 in position in 17 the assembly.
18 Completing the electrode cell in accordance with the 19 present invention are lead wires 92, 94 and 96 for connecting the testing electrode 90, counter electrode 86 21 and reference electrode 88 to controlled testing 22 potentials, a working pote~tials (or ground), and 23 reference potential.
24 As shown in FIG. 5, the electrical controls and circuits include a control amplifier 106 which controls 26 the current that flows between the counter electrode 86 27 and the working electrode 90 by means of the potential set 28 by the input and maintained between the reference 29 electrode 88 and the working electrode 90 via the feedback arrangement between the reference electrode 90, the 31 voltage follower 114, and the input to the control 32 amplifier 106. The thermodynamic state of the reference 33 electrode(s) will change in response to the changing pH of 34 the mobile phase gradient and the circuit will automatically keep the applied potential between the 36 reference and the working electrode to that set by the . .
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1 input signal thus effectively responding to the pH
2 gradient change of the mobile phase. This in turn keeps 3 the desired catalytic potential of the working electrode 4 at its optimal value throughout the gradient thus permitting compensation for the gradient elution changes 6 and also avoids the maintenance problems and leakage 7 problems typically associated with conventional glass 8 electrodes.
9 As should be clear from the foregoing, the electrochemical detection apparatus of the present 11 invention offers a number of advantages over prior art 12 electrochemical detectors.
13 It is to be appreciated that the invention is not 14 limited to application to liquid chromatography, but, rather the electrochemical detection apparatus may alsv be 1~ employed to monitor and/or measure the progress of a gas 17 chromatographic separation process. In this regard, in 18 some cases it may be possible to measure concentration or 19 constitutional changes in the gases directly. In other cases it will be necessary to carry out the method in the 21 presence of a liquid, preferably an electrolyte, e.g. by 22 dissolving the eluant gas ~rom the gas chromatography 23 apparatus in an electrolyte and passing the electrolyte 24 through the electrochemical detection cell.
Furthermore, the electrochemical detection apparatus 26 is not limited to use with chromatography separations, but 27 may also be advantageously employed for monitoring or 23 directly measuring a variety of sample solutions, for 29 example, of industrial, environmental, geophysical and biomedical interest. For example, the electrochemical 31 detection apparatus of the present invention may be 32 employed to provide on-line monitoring of a chemical 33 process flow stream or a public water supply system, or 34 for monitoring effluent from a sewage treatment facility~
Moreover, the electrochemical detection apparatus made 36 in accordance with the instant invention is not limited to .:~
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g 1 measuring only those compounds capable of undergoing 2 electrochemical reactions, but also is capable of 3 capacitive monitoring streaming solutions. For example, 4 the measuring electrochemically non-reactive materials, a repetitive pulse of short duration, e.g. 10 to 20 usec., 6 may be fed to current amplifier 100 and a capacitive spike 7 accumulated in a signal accumulator for a period of time, 8 e.g. 100 to 500 usec. prior to the calibration and 9 recording. In this way any substance capable of changing the capacitance of the electrode double-layer can be seen 11 at the signal output.

,

Claims

1. In an amperometric flow cell for electrochemically testing a sample solution, said cell comprising a sample test path having a testing electrode, a counter electrode and at least one reference electrode communicating therewith, the improvement wherein said reference electrode(s) comprises a solid state palladium electrode.

2. In a flow cell according to claim 1, wherein said counter electrode comprises palladium or palladium oxide.

3. A flow cell according to claim 2, wherein said counter electrode comprises a palladium foil electrode which defines the sample flow path at least in part.

4. A flow cell according to claim 1, wherein said reference electrode(s) consists of palladium.

5. A flow cell according to claim 1, wherein said reference electrode(s) consists of palladium oxide.

6. A flow cell according to claim 1, and including means for connecting said active testing electrode to controlled testing potentials, means for connecting said counter electrode to a counter potential or ground, and means for connecting said reference electrode(s) to a reference potential.

7. A flow cell according to claim 1, wherein said reference potential comprises an impedance network coupled in feedback arrangement to an amplifier whereby said reference electrode(s) may be actively driven in response to changes in the test solution.